Development of a stable semi-continuous lipid production system of an oleaginous Chlamydomonas sp. mutant using multi-omics profiling

Background Microalgal lipid production has attracted global attention in next-generation biofuel research. Nitrogen starvation, which drastically suppresses cell growth, is a common and strong trigger for lipid accumulation in microalgae. We previously developed a mutant Chlamydomonas sp. KAC1801, which can accumulate lipids irrespective of the presence or absence of nitrates. This study aimed to develop a feasible strategy for stable and continuous lipid production through semi-continuous culture of KAC1801. Results KAC1801 continuously accumulated > 20% lipid throughout the subculture (five generations) when inoculated with a dry cell weight of 0.8–0.9 g L−1 and cultured in a medium containing 18.7 mM nitrate, whereas the parent strain KOR1 accumulated only 9% lipid. Under these conditions, KAC1801 continuously produced biomass and consumed nitrates. Lipid productivity of 116.9 mg L−1 day−1 was achieved by semi-continuous cultivation of KAC1801, which was 2.3-fold higher than that of KOR1 (50.5 mg L−1 day−1). Metabolome and transcriptome analyses revealed a depression in photosynthesis and activation of nitrogen assimilation in KAC1801, which are the typical phenotypes of microalgae under nitrogen starvation. Conclusions By optimizing nitrate supply and cell density, a one-step cultivation system for Chlamydomonas sp. KAC1801 under nitrate-replete conditions was successfully developed. KAC1801 achieved a lipid productivity comparable to previously reported levels under nitrogen-limiting conditions. In the culture system of this study, metabolome and transcriptome analyses revealed a nitrogen starvation-like response in KAC1801. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02196-w.


Development of a lipid production method by the semi-continuous culture of KAC1801
This study aimed to develop a semi-continuous culture method for KAC1801 to achieve lipid production at levels feasible for commercialization. Light and nitrogen availability are important factors affecting cell growth and lipid accumulation in microalgae [7,8,15,16]. The impact of inoculation cell density and nitrate concentration during semi-continuous cultivation was examined. KAC1801 and the parental strain, KOR1, were subcultured every 24 h at an initial cell concentration of optical density at 750 nm (OD 750 ) = 0.5 (0.4-0.5 g L −1 ) or 1.0 (0.8-0.9 g L −1 ) in modified Bold's (MB) medium containing 9.3 mM (6 N) or 18.7 mM (12 N) NaNO 3 as the sole nitrogen source. Consistent with a previous report [24], biomass production and nitrate consumption by KAC1801 were lower, whereas lipid production was higher than that in KOR1 (Additional file 1: Fig. S1, Table S1). The mean lipid production in KAC1801 after 5 d of cultivation was 67.0 mg L -1 ( Table S1). Thus, maximal lipid production in KAC1801 was achieved in medium containing 12 N NaNO 3 at an initial cell density inoculation of OD 750 = 1.0, suggesting that these conditions are suitable for semi-continuous cultivation of KAC1801.
To evaluate the rate of lipid production in further detail, the semi-continuous culture of KAC1801 was performed for 5 d in MB 12 N medium by subculturing cells every 24 h at an inoculation cell density of OD 750 = 1.0 (Fig. 1). The values for biomass production, nitrate consumption, and lipid content during semi-continuous cultivation are summarized in Table 1. Biomass production in KAC1801 varied from 400.1 mg L -1 (day 4-day 5) to 610.6 mg L -1 (day 1-day 2), and that in KOR1 from 709.2 mg L -1 (day 2-day 3) to 925.1 mg L -1 (day 0day 1). Nitrate consumption by KAC1801 varied from 1.1 mM (day 4-day 5) to 3.0 mM (day 2-day 3), and that in KOR1 from 3.6 mM (day 1-day 2) to 6.1 mM (day 2day 3). The lipid content in KAC1801 was significantly greater than that in KOR1 during the entire cultivation period; KAC1801 constantly accumulated > 20% lipids, whereas the lipid content in KOR1 was < 9% throughout the cultivation period (Fig. 1c). Mean lipid production in KAC1801 was 116.9 mg L -1 day −1 , 2.3-fold greater than that in KOR1 (50.5 mg L -1 day −1 ).

Distribution of carbon in carbohydrates, proteins, and pigments
KAC1801 accumulated more lipids than KOR1 during semi-continuous culture, suggesting an altered carbon distribution resulting from CO 2 fixation. The levels of other major cell components in the microalga, including carbohydrates, proteins, and photosynthetic pigments, were also analyzed. In KAC1801, carbohydrate content, which is one of the major carbon storage forms in the model species of this study [6,25], was similar or slightly reduced relative to that in KOR1 (Fig. 2a). The protein and chlorophyll contents in KAC1801 were lower than that in KOR1 throughout the cultivation period (Fig. 2b, c). β-Carotene and lutein are the major carotenoids in the model species of this study [25]; the β-carotene content of KAC1801 was lower than that in KOR1 at almost all sampling points (Fig. 2d). In contrast, the lutein content of KAC1801 was similar or slightly lower than that in KOR1 (Fig. 2e). Thus, in contrast to the results for lipid accumulation, carbon distribution in proteins and photosynthetic pigments was lower in KAC1801 than in KOR1.

Identification of key metabolic changes involved in the altered carbon distribution in KAC1801
To identify the key metabolites in the altered carbon distribution phenotype, the metabolite pool size in KAC1801 from day 1 to 2 in semi-continuous culture was analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), according to the Calvin cycle, carbohydrate synthesis pathway, 2-C-methylerythritol 4-phosphate (MEP) pathway, glycolysis, lipid synthesis pathway, tricarboxylic acid (TCA) cycle, and nitrogen assimilation pathway (Fig. 3). In KAC1801, the pool sizes of sedoheptulose 7-phosphate (S7P) and 3-phosphoglycerate (3-PGA) were significantly lower in KAC1801 than in KOR1, while the differences in ribose 5-phosphate (R5P) and erythrose 4-phosphate (E4P) levels were not statistically significant (Fig. 4). These results are consistent with the low photosynthetic pigment content in KAC1801 (Fig. 2) and suggest lower levels of carbon fixation in KAC1801 than in KOR1. During glycolysis, the pool size of phosphoenolpyruvate (PEP) in KAC1801 was significantly lower than that in KOR1, while that of pyruvate (Pyr) and acetyl-CoA (AcCoA) were not different between these strains (Fig. 4). The levels of metabolites in the carbohydrate synthesis pathway, including fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), and ADP-glucose (ADP-glu), were similar between KOR1 and KAC1801 (Additional file 1: Fig. S3a-c), consistent with the results for carbohydrate content (Fig. 2a). In microalgae, carotenoid precursors (i.e., isopentenyl pyrophosphate and dimethylallyl diphosphate) are synthesized via the MEP pathway [26]. The pool sizes of the metabolites in the MEP pathway, including 1-deoxyd-xylulose 5-phosphate (DXP) and 2-C-methyld-erythritol-2,4-cyclopyrophosphate (MEcPP), were significantly lower in KAC1801 than in KOR1 (Additional file 1: Fig. S3d, e), suggesting a decrease in carbon flux for carotenoid synthesis in KAC1801. This result is consistent with the reduced β-carotene content in KAC1801 (Fig. 2d). In the TCA cycle, the pool sizes of citrate (Cit), malate (Mal), and fumarate (Fum) did not change in KAC1801 over time, except for a significant increase in Fum levels at 12 h (Fig. 4). In contrast, the pool sizes of 2-oxoglutarate (2-OG) and succinate  (Suc) were lower in KAC1801 than in KOR1. In nitrogen assimilation, NO 3 − is first converted to NH 4 + by nitrate reductase (NR) and nitrite reductase (NiR) [27]. In the glutamine synthase-glutamate synthase (GS/ GOGAT) cycle, glutamine (Gln) is synthesized by glutamine synthase (GS) using NH 4 + and glutamate (Glu) as substrates, and Gln and 2-OG are converted to Glu by glutamate synthase (GOGAT) [28]. The pool size of Glu in KAC1801 was significantly greater than that in KOR1 (Fig. 4).

C-turnover analysis of the dynamic metabolic flux in CO 2 fixation
Analysis of the dynamic changes in metabolite levels is important to understand the metabolic mechanism of the altered carbon distribution [25]. To evaluate metabolic profiles dynamically, the in vivo 13 C labeling of intracellular metabolites, newly synthesized from radiolabeled-CO 2 , was examined on day 1.5 over 10 min using cells in semi-continuous culture (Fig. 5). In the Calvin cycle, the 13 C fraction of S7P and 3-PGA of KAC1801 was lower than that in KOR1, suggesting that CO 2 fixation was decreased in KAC1801. In glycolysis, which occurs downstream of the Calvin cycle, there was no difference in the 13 C fraction of PEP, and that of Pyr was lower in KAC1801 than in KOR1. The 13 C fraction of metabolites related to lipid synthesis, including AcCoA and G3P, was lower in KAC1801 than in KOR1. In the TCA cycle, the 13 C fraction of Cit and Mal was lower in KAC1801, while that of 2-OG, Suc, and Fum was similar in these strains.

Identification of key genes differently expressed in KAC1801
To elucidate the underlying mechanism of the altered carbon distribution in KAC1801 at the transcript level, comprehensive gene expression profiling was performed via RNA-seq analysis using cells harvested at day 1.5. In total, 899 genes were identified as differently expressed genes (DEGs) between KOR1 and KAC1801. Among these, 275 and 624 genes were downregulated and upregulated in KAC1801, respectively (Additional file 2). Gene ontology (GO) analysis of the DEGs indicated that 22 and 12 categories were downregulated and upregulated in KAC1801, respectively (Fig. 6).

Discussion
In microalgae, nitrogen starvation not only is a strong trigger for lipid accumulation but also suppresses cell growth [17], which increases the risk of predation by environmental contaminants. Several microalgal strains have been developed to accumulate lipids under growth conditions [21][22][23][24]; however, a culture method to fully utilize the potential of this technology had not been established. Chlamydomonas sp. KAC1801 is a mutant which accumulates a high level of lipids under nitratereplete conditions [24]. Using KAC1801, the present study achieved stable and continuous lipid production in a semi-continuous nitrate-replete culture system. KAC1801 produced 116.9 mg L −1 day −1 lipids (Table 4), which is comparable to the levels produced in previous semi-continuous and nitrogen-limited cultivation studies using microalgae strains that accumulated lipids under nitrogen starvation. For example, Han et al. and Hsieh and Wu achieved a lipid productivity of 115 and 139 mg L −1 day −1 in NaNO 3 -(~ 2.4 mM) and urea-limiting conditions (~ 0.5 mM), respectively [18,19]. The present study developed a simple one-step semi-continuous cultivation method for biofuel production using a mutant strain that accumulated lipids under nitrate-replete conditions (> 11.8 mM NaNO 3 ).
In general, the photosynthetic pigment content as well as the ratio of nitrogen-containing compounds, such as proteins and chlorophylls, decrease under nitrogendeficient conditions [31][32][33][34]. Under nitrogen-deficient conditions, chlorophylls and β-carotene decreased in Chlamydomonas sp. strains [25]. In the present study, both photosynthetic pigments (chlorophylls and β-carotene) and proteins decreased in KAC1801 compared to KOR1 (Fig. 2). Decreased protein content has been reported in a Nannochloropsis mutant grown under nutrient-replete conditions in which it can accumulate lipids [21]. Because nitrate consumption in KAC1801 was significantly lower than that in KOR1 (Fig. 1b), it was hypothesized that the intracellular level of nitrogen was decreased in KAC1801, which consequently induced nitrogen starvation-like responses, i.e., the accumulation of lipids and a decrease in photosynthetic pigments and proteins. The decrease in β-carotene content in KAC1801 may be due to lowered carbon flux for carotenoid synthesis, which was supported by the data of decreased pool sizes in the MEP pathway (Additional file 1: Fig. S3). The present study revealed that 2-OG and Suc decreased and Glu increased in KAC1801 (Fig. 4), suggesting increased GS activity. This may also be a part of the nitrogen starvation-like response because upregulation of GS and GOGAT genes under nitrogen-deficient conditions was reported in Nannochloropsis [35]. In KAC1801, the transcript levels of genes related to photosynthesis, for example, light harvesting (LhcI-2, LhcI-3, LHCA2, LHCA9, LHCB4, and lhcb5) and carbon fixation (Rca and SEBP1), were decreased (Fig. 6, Additional file 1: Table S2). These genes are known to be downregulated in Chlamydomonas reinhardtii and Dunaliella tertiolecta under nitrogen-deficient conditions [36,37]. The CYN38 gene, which contributes to the assembly and repair of photosystem II [38], was downregulated in KAC1801. This gene was also reported as a downregulated gene under nitrogen starvation conditions in C. reinhardtii [39]. In addition, pool size and turnover rate of metabolites in the Calvin cycle, including 3-PGA and S7P, were lower in KAC1801 (Figs. 4, 5). These results suggest that photosynthetic activity, especially light harvesting and carbon fixation, was lower in KAC1801, which may explain the reduced biomass production (Fig. 1a). KAC1801's reduced photosynthetic activity may be due to decreased chlorophyll content (Fig. 2c), but this is uncertain because the mutant was created through random mutagenesis and thus may harbor mutations unrelated to pigment accumulation [24]. This study proposes that the nitrogen starvation-like response in KAC1801 was the cause of increased lipid accumulation under the nitrogen-replete conditions. The transcript levels of genes involved in the TCA cycle and glyoxylate shunt (i.e., ICL1, MS1, MDH2, and CIS2) were higher in KAC1801 (Additional file 1: Fig.  S4). This suggests enhancement of the glyoxylate cycle in KAC1801, which is advantageous for preventing emission of carbon sources because the CO 2 -producing reactions involved in the TCA cycle are bypassed by the glyoxylate shunt.
Lipid content was considerably higher in KAC1801 than in KOR1 (Fig. 1c), though the pool sizes and 13 C fractions of the lipid precursors as well as the expression levels of genes related to lipid synthesis were similar or decreased between groups (Figs. 4, 5). Mutational analysis of KAC1801 was performed to identify genes responsible for the lipid accumulation, and mutations in 811 coding sequences were determined (data not shown). However, most of the identified genes were functionally uncharacterized, and no responsible gene was determined. Although further studies are required to elucidate the direct mechanism of lipid accumulation in KAC1801 under nitrate-replete conditions, it is hypothesized that KAC1801 may show enhanced lipid synthesis and decreased lipid degradation. For example, overexpression of the G3P acyltransferase GPAT1 isoform in Cyanidioschyzon merolae increased lipid productivity by 56.1fold without inhibiting growth [22]. In C. reinhardtii, genes encoding diacylglycerol acyltransferases (DGAT1 and DGAT2), phospholipid:diacylglycerol acyltransferase (PDAT), and lysophosphatidic acid acyltransferase (LPAAT ), which contribute to lipid synthesis, were upregulated under nitrogen-deficient conditions [40,41]. In addition, knockout of the gene encoding phospholipase A 2 , which contributes to lipid degradation, improved lipid productivity in C. reinhardtii under growth conditions [42].

Conclusions
This study describes a method for stable lipid production in the semi-continuous cultivation of Chlamydomonas sp. KAC1801 under nitrate-replete conditions by optimizing nitrate supply and cell density. KAC1801 constantly accumulated lipids at > 20% of DCW during 5 d of semi-continuous cultivation and achieved a lipid productivity of 117 mg L −1 day −1 , which was comparable to previously reported levels of productivity under nitrogen-limiting conditions. Metabolome and transcriptome analyses revealed a nitrogen starvation-like response in KAC1801. Additionally, this one-step microalga lipid production method provides insights into the molecular responses associated with semi-continuous lipid production under nitrate-replete conditions.

Strains and culture conditions
Cultivation of microalgae, Chlamydomonas sp. KAC1801 [24] PO 4 , and 0.43 mM NaCl) and trace elements as described in a previous report [43], including 2% (w/v) sea salt (Sigma-Aldrich, St. Louis, MO, USA). The CO 2 concentration was adjusted to 2% by adding 50 mL of 2 M K 2 CO 3 /KHCO 3 solution to the lower stage. After pre-cultivation for 5 days, the optical density at 750 nm (OD 750 ) was measured using a UV mini-1240 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). For semi-continuous cultivation, the cells were inoculated into new flasks every 24 h with an initial OD 750 of 1.0.

Measurement of biomass production
The culture broth was centrifuged at 8000×g for 1 min and washed once with ultrapure water. The cell pellet was lyophilized using an FDU-1200 (Tokyo Rikakikai, Tokyo, Japan). Daily biomass production (mg L −1 ) during semi-continuous cultivation was calculated as BC x (mg L −1 ) − BC y (mg L −1 ), where BC x is the biomass concentration after 24 h of inoculation and BC y is the biomass concentration after 0 h of inoculation.

Measurement of nitrate concentration
The culture broth was centrifuged at 8000×g for 1 min. The absorbance of the supernatant was measured at 220 nm to determine the nitrate concentration using a calibration curve [44]. Daily nitrate consumption during semi-continuous cultivation was calculated using the following formula: Nitrate consumption (mM) = NC x (mM) -NC y (mM), where NC x is the nitrate concentration after 0 h of inoculation, and NC y is the nitrate concentration after 24 h of inoculation.

Lipid analysis
Cells were harvested by centrifugation at 8000×g for 1 min, washed once with ultrapure water, and lyophilized. Lyophilized cells ( . Heptadecanoic acid (Sigma-Aldrich) was used as an internal standard for the quantification of fatty acids. The lipid content was calculated as the total intracellular fatty acid content per DCW [24,25]. Daily lipid production (mg L −1 ) during semi-continuous cultivation was calculated as where BC x is the biomass concentration after 24 h of inoculation, LC x is the lipid content after 24 h of inoculation, BC y is the biomass concentration after 0 h of inoculation, and LC y is the lipid content after 0 h of inoculation.

Carbohydrate analysis
Lyophilized cells (2-3 mg) were suspended in 2 mL of 4% (v/v) sulfuric acid and autoclaved at 120 °C for 30 min. The solution was neutralized by adding 1 mL of 22% (w/v) sodium carbonate. Cell debris was removed by centrifugation at 10,000×g for 10 min and subsequent filtration using a Shim-pack SPR-Pb column (Shimadzu). The glucose concentration was determined using a highperformance liquid chromatography (HPLC) system (Shimadzu) equipped with an Aminex HPX-87H column (9 μm, 300 mm × 7.8 mm; Bio-Rad Laboratories, Hercules, CA, USA). Soluble starch (CAS number: 9005-84-9, Nacalai Tesque) was used as the quantitative standard. The carbohydrate content was determined using a calibration curve [25].

Protein analysis
Lyophilized cells (2-3 mg) were suspended in 0.2 mL of 1 N NaOH and incubated at 80 °C for 10 min. Subsequently, 1.8 mL of water was added and the solution was centrifuged at 12,000×g for 30 min. The protein concentration in the supernatant was analyzed using a Takara BCA protein assay kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions [45,46].

Pigment analysis
Lyophilized cells (2-3 mg) were suspended in 500 μL of methanol:acetone (5:5 [v/v]) and fractured using 0.5 mm glass beads in a multi-bead shocker MB1001C(S) as described for lipid analysis. The samples were centrifuged at 10,000×g for 2 min at 4 °C, and the supernatant was transferred to a new microtube. The extraction procedure was repeated four times to obtain 2 mL of supernatant. The supernatant (330 μL) was dried in a vacuum using an evaporator CEV-3100 (EYELA, Tokyo, Japan), resuspended in 500 μL of chloroform:acetonitrile ( [25,47].

Metabolome analysis
Cells equivalent to 5 mg DCW were harvested using 10 µm pore size filters (Merck Millipore, Burlington, MA, USA), washed once with 20 mM ammonium carbonate, and immediately suspended in 1 mL of pre-cooled (-30 °C) methanol containing 36 µM piperazine-1,4-bis (2-ethanesulfonic acid) (Dojindo Laboratories, Kumamoto, Japan) and 36 µM l-methionine sulfone (Sigma-Aldrich) as internal standards. The suspension (500 µL) was subjected to cell disruption using 0.5 mm glass beads YGB05 in a multi-bead shocker MB1001C(S) as described for lipid analysis. Subsequently, 150 µL of chloroform and 50 µL of ultrapure water were added and mixed by vortexing for 10 s. After centrifugation at 14,000×g for 5 min at 4 °C, 400 µL of supernatant was collected, mixed with 200 µL of ultrapure water by vortexing for 10 s, and centrifuged at 14,000×g for 5 min at 4 °C. The upper phase was filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit UFC5003BK (Merck Millipore) at 14,000×g for 50 min at 4 °C. The flow-through (300 μL) was dried in a vacuum using an evaporator CEV-3100 (EYELA). Dried samples were resuspended in 20 µL of ultrapure water and analyzed by CE-TOFMS using a G7100 CE and G6224AA liquid chromatograph/mass selective detector (LC/MSD) TOF system (Agilent Technologies) [25,48].

Dynamic metabolome analysis
To perform in vivo 13 C labeling of newly synthesized metabolites using radiolabeled CO 2 , cells were harvested on day 1.5 of semi-continuous culture using 10 µm pore size filters (Merck Millipore) and resuspended in MB 12 N medium containing 2% (w/v) sea salt and 25 mM NaH 13 CO 3 (Cambridge Isotope Laboratories, Tewksbury, MA, USA). After incubation under white fluorescent lamps at 250 μmol photons m −2 s −1 and shaking at 100 rpm, cells were harvested and the intracellular metabolites were analyzed as described for the metabolome analysis. The 13 C labeling ratio was calculated as described in a previous report [25,48].

Genome analysis
The whole genome sequence of Chlamydomonas sp. was determined using the hybrid assembly method and Nanopore and Illumina KOR1 reads from a previous study [25]. Briefly, low-quality regions in the Nanopore long-reads were trimmed using Yanagiba v. 1.0.0 and assembled using Canu v. 1.7 [49]. Genome mapping analysis against the resulting assembly was performed using Burrows-Wheeler Aligner (BWA) v. 0.7.12 [50] and Illumina sequence reads; assembly polishing was performed using Pilon v. 1.23 against Illumina mapping data (https:// github. com/ broad insti tute/ pilon). Prediction of gene coding sequences was performed using AUGUS-TUS software v. 3.3.3 and a training set for C. reinhardtii (NCBI: txid3055) [51]. Functional assignments of the predicted genes were based on a BLASTP homology search using an E-value cutoff of 1e −5 against the previously reported C. reinhardtii genome [52]. The sequencing data obtained here were used as the reference in the RNA-seq analysis described below.

Transcriptome analysis
Cells were harvested on day 1.5 of the semi-continuous culture by centrifugation at 12,000×g for 1 min, immediately frozen in liquid nitrogen, and stored at -80 °C. Total RNA was extracted using an RNeasy Plus Universal Kit (Qiagen, Tokyo, Japan), according to the manufacturer's instructions. RNA integrity was determined using an Agilent Bioanalyzer 2100 and Agilent RNA 6000 Nano Kit (Agilent Technologies). Using a NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), library preparation was performed using 500 ng of total RNA according to the manufacturer's protocol, with 12 cycles of polymerase chain reaction (PCR). The library concentration and quality were assessed using an Agilent DNA 1000 Kit and the Agilent Bioanalyzer 2100 (Agilent Technologies). The library concentration was determined using a KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, DE, USA) and confirmed using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, CA, USA). The cDNA library was sequenced using an Illumina NextSeq 500 platform, yielding 150 bp paired-end